This invention relates to bioherbicides for controlling cyanobacteria, and more particularly, to a new bacterium and method of use thereof for the biological control of a variety of pest cyanobacteria that cause an off-flavor in water or aquatic life, such as fish, living in the water.
Off-flavor has been identified as one of the most serious problems in the catfish industry. Off-flavor can render fish unpalatable, and thus delay the harvesting and marketing of commercially produced fish such as channel catfish. Losses due to off-flavor often run into the thousands of dollars for each incidence of the problem, and annual losses exceed $100 million.
Off-flavor is a complex phenomenon that is related to the absorption of compounds that are produced by cyanobacteria, algae, and actinomycetes. Geosmin (trans-1,10-dimethyl-trans-9-decalol) and 2-methylisoborneol (MIB) are produced by cyanobacteria and actinomycetes, and these two compounds have been shown to cause off-flavor in channel catfish. Although the roles of the various organisms and compounds are not completely understood, most geosmin off-flavor in commercially produced channel catfish has been attributed to species of Anabaena, while MIB off-flavor has been linked to a planktonic species identified as Oscillatoria cf. chalybea. 
The concept of controlling off-flavor by management of cyanobacteria has been proposed (Van der Ploeg, M.; Proc. World Aquaculture Soc., p. 71, 1994 (Abst.)), but no selective control measures have been developed. Conventional control practices include application of broad-spectrum algicides, such as copper sulfate or diurion [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. The introduction of certain chemical agents to fresh water reservoirs or fisheries for the reduction, control or elimination of off-flavor can be undesirable. Alternative approaches not requiring the addition of chemical agents are preferred but almost nonexistant.
Viruses and microorganisms are involved in the population dynamics of cyanobacteria in nature, but there are no reports of attempts to use biological control technology to manage cyanobacteria in channel catfish ponds.
Thus, there remains a significant need for the development of alternate strategies for the reduction of off-flavor in fresh water supplies and in fish harvested from fresh water nurseries.
It has now been found that the novel bacterium of the invention is effective in controlling the susceptible species of cyanobacteria. In addition, the novel bacterium can control, reduce or eliminate the incidence of organisms responsible for off-flavor in commercially produced fish and fresh water, can manage the water quality of aquaculture and water supplies, and can be used in treating municipal or industrial waste.
Accordingly, one aspect of the present invention provides an isolated form of a pathogenic bacterium, or progeny thereof, capable of controlling the growth of at least one cyanobacterium, the pathogenic bacterium having a 16S rRNA gene sequence substantially homologous to that of SEQ. ID No 1.
The method of the invention employs a previously unknown pathogenic bacterium such as the Gram negative rod Bacterium SG-3 which exhibits flagellar motility. The Bacterium SG-3 has been deposited in the ARS Patent Culture Collection and assigned the number NRRL B-30043. RNA sequence data suggests a possible phylogenetic relation to the Xanthomonas spp.
Another aspect of the invention provides a method of reducing or eliminating off-flavor in water, fish, or other aquatic life forms in water. The method involves treating water with a cyanobacterial reducing or eliminating amount of a pathogenic bacterium or progeny thereof for a period of time sufficient to reduce or eliminate cyanobateria in the water thereby reducing or eliminating off-flavor in the water.
The pathogenic bacterium is preferably Bacterium SG-3. The off-flavor in the water, fish or other aquatic life form are usually caused by algae, cyanobacteria, undesired bacteria, or one or more metabolites, products or chemical agents thereof. In a preferred embodiment of the present invention, the method also involves preparing an aqueous suspension comprising Bacterium SG-3 or progeny thereof prior to treating the water with Bacterium SG-3.
The pathogenic bacterium is preferably present in preparations including a suspension, powder, solid, slug, freeze-dried powder, pellet, granule, cell lysate, homogenate, concentrate, diluted solution, or immobilized cell preparation. When granules containing the pathogenic bacterium are used as inoculum, the granules can be produced, for example, by pelletization of a suspension containing the bacterium. An exemplary pelletization procedure is described herein and in U.S. Pat. No. 5,739,019, the disclosure of which is hereby incorporated by reference in its entirety. These preparations of Bacterium SG-3 are suitable for corrective and preventitive applications.
When present in a composition, the pathogenic bacteria-containing composition can be formulated as a granule, spray dried solid, freeze dried solid, emulsifiable concentrate, wettable powder, dust aerosol, dry flowable powder or any other conventional formulation used in the application of algicides or herbicides to bodies of water. The Bacterium SG-3 is used in its native form as obtained from a culture or in a genetically engineered form. The genetically engineered form will preferably exhibit improved efficacy, enhanced pathogenicity, improved storage and formulation properties or enhanced host range.
The pathogenic bacterium may produce a lytic agent that can lyse a cyanobacterium, alga or undesired bacterium. The lytic agent could be a protein or chemical agent.
Another aspect of the invention provides a method for reducing or eliminating off-flavor in an aqueous waste or aquaculture. According to this aspect of the invention, the method involves inoculating aqueous waste or aquaculture with an inoculum comprising an off-flavor reducing or eliminating amount of Bacterium SG-3 or progeny thereof to form a mixture and incubating the mixture to increase a cellular titre of the bacterium in the mixture and to reduce or eliminate the off-flavor.
Yet another aspect of the invention provides a method of controlling an amount of cyanobacteria, algae or undesired bacteria in a water-containing environment. According to this aspect of the invention, the method involves treating cyanobacteria, algae or undesired bacteria with an effective amount of Bacterium SG-3 or progeny thereof for a period of time sufficient to control the amount of cyanobacteria, algae or undesired bacteria in the water-containing environment.
In addition to cyanobacteria and algae, susceptible viruses, fungi and bacteria can also be controlled by using Bacterium SG-3 in combination with chemicals, herbicides, other pathogenic agents or salt. Thus, still another aspect of the invention is a method for controlling cyanobacteria and Plectonema spp. in water by treating a body of water with an effective amount of Bacterium SG-3 and Cyanophage LPP-1 for a period of time sufficient to control the cyanobacteria and Plectonema spp. The artisan of ordinary skill will understand that the pathogenic bacterium of the invention is used to control other susceptible pest bacteria in water, fish or other aquatic life forms. Other susceptible pest bacteria include one or more of Edwardsiella spp., Flexibacter spp., Aeromonas spp., and Pseudomonas spp., in particular, Edwardsiella ictaluri, Flexibacter columnaris, Aeromonas salmonicida, A. hydrophila, and A. sobria. 
A composition comprising a combination of a pathogenic bacterium and one or more other pathogenic organisms can provide control of a broader range of cyanobacteria and algae than is achieved using either alone. Thus, the present invention also provides an anti-algae or anti-cyanobacterial composition for the control of susceptible algae or susceptible cyanobacteria comprising a pathogenic bacterium, such as Bacterium SG-3 and a pathogenic virus, such as Cyanophage LPP-1 which is pathogenic toward Plectonema spp.
Another aspect of the present invention provides a method of producing the Bacterium SG-3. In a first method, the Bacterium SG-3 of the invention was isolated from a fresh water pond according to the methods of Example 1. In a second method, Bacterium SG-3 was produced by culturing in the exemplary growth medium described in Example 2. Bacterium SG-3 can also be harvested from bodies of water containing the bacterium.